Peptide suppressing IκBα phosphorylation

ABSTRACT

The present invention discloses an antiinflamatory agent or immunosuppressant that contains as its active ingredient a peptide that inhibits phosphorylation of IκBα, an example of which is the peptide having the following amino acid sequence (SEQ ID NO: 1): Met-Leu-Pro-Glu-Ser-Glu-Asp-Glu-Glu-Ser-Tyr-Asp-Thr-Glu-Ser-Glu-Phe-Thr-Glu-Phe-Thr-Glu-Asp-Glu-Leu.

TECHNICAL FIELD

The present invention relates to a peptide capable of inhibitingphosphorylation of IκBα, and more particularly, to a peptide useful asan antiphlogistic or immunosuppressant.

BACKGROUND ART

A region referred to as κB and usually composed of 10 nucleotides wasclearly shown to promote gene transcription activity as a part of anenhancer by analysis of the protein-binding region in the enhancerregion of human immunodeficiency virus (HIV) and antibody κ light chain.When the transcription regulating protein, nuclear factor κB (NFκB)binds to this region, transcription of gene downstream from the κBregion is promoted (Gilmore, T. & Morin, P. J.: Trends in Genetics(1993) 9, 427-432).

According to a study using a pre-B cell line, 70Z/3 cells, and Helacells, it is known that these cells contain in their cytoplasm NFκB thatdoes not have any DNA binding activity, and that by phorbol esterstimulation, the DNA binding activity is derepressed causing NFκB totranslocate into the nucleus (Baeuerle, P. A. & Baltimore, D.: Cell(1988) 53, 211-217).

NFκB is a heterodimeric or homodimeric transcription factor comprisingtwo subunits (p50, p65), and is induced by various stimuli. It iscurrently estimated to be involved in the transcription activity of morethan 20 genes (Baeuerle, P. A.: Biochim. Biophys. Acta (1991) 1072;63-80).

NFκB is also located upstream from the cytokine genes of Interleukin-8,Interleukin-1β, tumor necrosis factor (TNF)-α, Interleukin-6 andInterleukin-2 as well as cyclooxygenase-2 (Kujubu, D. A. et al., J.Biol. Chem. (1991) 266, 12866-12872) or 5-lipoxygenase (Hoshiko, S. etal., Proc. Natl. Acad. Sci. U.S.A. (1990) 87, 9073-9077; Chopra, A. etal., Biochem. Biophys. Res. Commun. (1992) 185, 489-495), which areenzymes involved in the biosynthesis of metabolites of the arachidonicacid cascade, mediators of inflammations, and the genes of adhesionfactors such as ELAM-1 (endothelial leukocyte adhesion molecule-1)(Hooft van Huijsduijnen, R. et al., J. Biol. Chem. (1992) 267,22385-22391) and VCAM-1 (Vascular Cell Adhesion Molecule-1) (Iademarco,M. F. et al., J. Biol. Chem. (1992) 267, 16323-16329). NFκB isconsidered to be involved in the control of the transcription of thesegenes.

The action of steroids, which are currently used as antiinflammatoryagents (Mukaida, N. et al., J. Biol. Chem. (1994) 269, 13289-13295) andthe powerful immunosuppressant, FK-506 (Okamoto, S.-i. et al., J. Biol.Chem. (1994) 269, 8582-8589) is suggested to at least involve aninhibition of the activation of NFκB. In the cytoplasm, NFκB binds withprotein IκBα, which inhibits the function of NFκB, resulting ininhibition of its activation (Inoue, J-I. et al., Proc. Natl. Acad. Sci.USA (1992) 89, 4333-4337; Hatada, E. et al., EMBO (1993) 12, 2781-2788).

Zabel, U. et al. reported to have isolated IκBα as a 37 kDa homogeneousprotein from human placenta (Cell (1990) 61, 255-265). This was followedby the isolation of the cDNA of human IκBα (Sporn, S. A. et al., J.Immunol. (1990) 144, 4434-4441; Haskill, S. et al., Cell (1991) 65,1281-1289, see WO 92-20795).

IκBα not only inhibits the binding of NFκB to DNA, but also inhibits thetranslocation of NFκB into the nucleus (Baeuerle, P. A. & Baltimore, B.Science (1988) 242, 540-546). IκBα has been clearly shown to bephosphorylated in response to various stimuli such as TNFα and so forth.

NFκB translocates into the nucleus and binds to the protein-binding siteon DNA as a result of phosphorylation or degradation of IκBα in thismanner or by phosphorylation of the proteins comprising NFκB, followedby activation of the transcription of genes located downstream(Baeuerle, P. A.: Biochim. Biophys. Acta (1991) 1072, 63-80). Althoughthe above-mentioned mechanism is known to occur at the time ofactivation of NFκB, the enzyme group that phosphorylates NFκB and IκBαin vivo, the phosphorylation site and its detailed mechanism have notbeen determined.

DISCLOSURE OF THE INVENTION

So far steroids and other drugs have been used as antiinflammatoryagent. Steroids have the risk of inducing adverse side effects includingsusceptibility to infection, diabetes and cataracts. Although nucleicacid synthesis inhibitors and similar drugs have been used asimmunorepressors, these result in adverse side effects such asinhibition of bone marrow function, liver injury, susceptibility toinfection and digestive tract disorders. Thus, there has been a desireto develop a drug that does not produce these types of adverse sideeffects.

The object of the present invention is to provide a peptide thatinhibits phosphorylation of IκBα, comprised of the phosphorylation siteof IκBα, for specifically inhibiting the gene expression of inflammatorycytokines and inflammatory mediators as well as the release of theirgene products.

During the course of studying the mechanism of the activation of NFκB,the inventors of the present invention succeeded in identifying thephosphorylation site of IκBα by using various types of partially deletedmutants and substituted mutants of IκBα. Moreover, by placing a peptidecorresponding with said phosphorylation site in the presence of IκBα, itwas found that the phosphorylation of IκBα was inhibited, therebyinhibiting the activation of NFκB.

Thus, the present invention provides a peptide having an amino acidsequence corresponding to the phosphorylation site of IκBα, and itspharmacologically acceptable salts.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates plasmid pGST-MD containing theglutathione-S transferase (GST) gene into which a DNA coding for thefull length of human IκBα has been introduced.

FIG. 2 schematically illustrates constructed fused proteins of varioustypes of partially deleted IκBα mutants with GST.

FIG. 3 schematically illustrates the amino acid sequences of the acidicregions present at the C-terminus of IκBα (SEQ ID NO: 19) andsubstituted IκBα mutant (SEQ ID NO: 20). Each symbol is an amino acidrepresented using single letter.

FIG. 4 is a diagram of electrophoresis of the phosphorylation of GSTfused proteins of the full length of IκBα (wt) as well as deletedmutants del-9, del-10, del-1, del-5 and del-4 as determined by in vitrokinase assay in the solid phase.

FIG. 5 is a diagram of electrophoresis of the phosphorylation of GSTfused proteins of the full length of IκBα (wt) and deleted mutantsdel-9, del-10 and del-1 and a substituted mutant (mt-K) as determined byin vitro kinase assay in the liquid phase.

FIG. 6 illustrates the phosphorylation state of IκBα in an in vitrokinase assay in the case wherein the peptide corresponding with thephosphorylation site of IκBα (IκBα kinase substrate) and unrelatedpeptides which are casein kinase substrate (CKII substrate) and MAPkinase substrate (MAPK substrate) were added. The phosphorylation ofeach treatment when that of the control (peptide non-addition group) istaken to be 100% is expressed as a relative ratio (n=3).

FIG. 7 illustrates the degree of phosphorylation of each aminoacid-substituted peptide caused by the partially purified IκBαphosphorylase (kinase). The degree of phosphorylation of each peptideaddition group when that of the positive control (group to which wasadded the peptide correspondig with the phosphorylation site of IκBα) istaken to be 100% is expressed as a relative ratio.

FIG. 8 illustrates a gel shift assay wherein the peptide correspondingwith site of IκBα (IκBα kinase substrate) and control peptides which area casein kinase substrate (CKII substrate) and MAP kinase substrate(MAPK substrate) were added.

FIG. 9 illustrates the analysis and quantitative determination of a gelshift assay using an image analyzer in the case wherein the peptide ofthe phosphorylation site of IκBα (IκBαK) and control peptides consistingof casein kinase substrate (CKII substrate) and MAP kinase substrate(MAPK substrate) were added. The relative ratio of each treatment groupis shown when that of the LPS(+)/peptide non-addition group is taken tobe 100% (n=4).

DETAILED DESCRIPTION

As is clear from Example 1 (2), the phosphorylation site of IκBα issurmised to be Ser (288), Thr (291) and Ser (293) in the sequence Ser(288)-Tyr (289)-Asp (290)-Thr (291)-Glu (292)-Ser (293)-Glu (294)(residues 10-16 of SEQ ID NO: 1) within the amino acid sequence of saidprotein. Thus, the peptide in the present invention that has the aminoacid sequence corresponding with the phosphorylation site of IκBα and isable to inhibit the phosphorylation of IκBα is that consists of theamino acid sequence Ser-Tyr-Asp-Thr-Glu-Ser (residues 10-15 of SEQ IDNO: 1) (referred to as the core amino acid sequence), or comprises thiscore amino acid sequence.

Comprising this amino acid sequence refers to that in which theN-terminal side or C-terminal side, or both sides of the amino acidsequence are extended with amino acid or a peptide. The amino acid orpeptide that extends the N-terminal and/or C-terminal may be any aminoacid or peptide provided it inhibits phosphorylation of IκBα.

However, according to a preferable embodiment of the present invention,the amino acid or peptide that extends the N-terminal side and/orC-terminal side of the above-mentioned core amino acid sequence is thatwhich is present in the amino acid sequence of IκBα and is adjacent tothe above-mentioned core amino acid sequence. This means that the wholepeptide composed of the core amino acid sequence and the terminal aminoacid or amino acid sequence that extends it forms a portion of the aminoacid sequence of IκBα.

If the amino acid sequence within IκBα isMet(279)-Leu(280)-Pro(281)-Glu(282)-Ser(283)-Glu(284)-Asp(285)-Glu(286)-Glu(287)-Ser(288)-Tyr(289)-Asp(290)-Thr(291)-Glu(292)-Ser(293)-Glu(294)-Phe(295)-Thr(296)-Glu(297)-Phe(298)-Thr(299)-Glu(300)-Asp(301)-Glu(302)-Leu(303)(SEQ ID NO: 1), an example of a peptide having this amino acid sequencecomprises a portion of this sequence and has the following N-terminalsand C-terminals:

Ser(288)-Glu(²⁹⁴), Ser(288)-Phe(295), Ser(288) -Thr(296),Ser(288)-Glu(297), Ser(288)-Phe(298), Ser(288)-Thr(299),Ser(288)-Glu(300), Ser(288)-Asp(301), Ser(288)-Glu(302),Ser(288)-Leu(303),

Glu(287)-Glu(294), Glu(287)-Phe(295), Glu(287)-Thr(296),Glu(287)-Glu(297), Glu(287)-Phe(298), Glu(287)-Thr(299), Glu287)-Glu(300), Glu(287)-Asp(301), Glu(287)-Glu(302), Glu(287)-Leu(303),

Glu(286)-Glu(294), Glu(286)-Phe(295), Glu(286)-Thr(296),Glu(286)-Glu(297), Glu(286)-Phe(298), Glu(286)-Thr(299),Glu(286)-Glu(300), Glu(286)-Asp(301), Glu(286)-Glu(302),Glu(286)-Leu(303), Asp(285)-Glu(294), Asp(285)-Phe(295),Asp(285)-Thr(296), Asp(285)-Glu(297), Asp(285)-Phe(298),Asp(285)-Thr(299), Asp(285)-Glu(300), Asp(285)-Asp(301),Asp(285)-Glu(302), Asp(285)-Leu(303),

Glu(284)-Glu(294), Glu(284)-Phe(295), Glu(284)-Thr(296),Glu(284)-Glu(297), Glu(284)-Phe(298), Glu(284)-Thr(299),Glu(284)-Glu(300), Glu(284)-Asp(301), Glu(284)-Glu(302),Glu(284)-Leu(303),

Ser(283)-Glu(294), Ser(283)-Phe(295), Ser(283)-Thr(296),Ser(283)-Glu(297), Ser(283)-Phe(298), Ser(283)-Thr(299),Ser(283)-Glu(300), Ser(283)-Asp(301), Ser(283)-Glu(302),Ser(283)-Leu(303),

Glu(282)-Glu(294), Glu(282)-Phe(295), Glu(282)-Thr(296),Glu(282)-Glu(297), Glu(282)-Phe(298), Glu(282)-Thr(299),Glu(282)-Glu(300), Glu(282)-Asp(301), Glu(282)-Glu(302),Glu(282)-Leu(303),

Pro(281)-Glu(294), Pro(281)-Phe(295), Pro(281)-Thr(296),Pro(281)-Glu(297), Pro(281)-Phe(298), Pro(281)-Thr(299),Pro(281)-Glu(300), Pro(281)-Asp(301), Pro(281)-Glu(302),Pro(281)-Leu(303),

Leu(280)-Glu(294), Leu(280)-Phe(295), Leu(280)-Thr(296),Leu(280)-Glu(297), Leu(280)-Phe(298), Leu(280)-Thr(299),Leu(280)-Glu(300), Leu(280)-Asp(301), Leu(280)-Glu(302),Leu(280)-Leu(303),

Met(279)-Glu(294), Met(279)-Phe(295), Met(279)-Thr(296),Met(279)-Glu(297), Met(279)-Phe(298), Met(279)-Thr(299),Met(279)-Glu(300), Met(279)-Asp(301), Met(279)-Glu(302),Met(279)-Leu(303).

The following are examples of peptides having an amino acid sequence asdescribed above.

Peptides containing the amino acid sequence:Met-Leu-Pro-Glu-Ser-Glu-Asp-Glu-Glu-Ser-Tyr-Asp-Thr-Glu-Ser-Glu-Phe-Thr-Glu-Phe-Thr-Glu-Asp-Glu-Leu(SEQ ID NO: 1);

Peptides containing the amino acid sequence:Met-Leu-Pro-Glu-Ser-Glu-Asp-Glu-Glu-Ser-Tyr-Asp-Thr-Glu-Ser-Glu-Phe (SEQID NO: 2);

Peptides containing the amino acid sequence:Ser-Glu-Asp-Glu-Glu-Ser-Tyr-Asp-Thr-Glu-Ser-Glu-Phe (SEQ ID NO: 3); and

Peptides containing the amino acid sequence: Ser-Tyr-Asp-Thr-Glu-Ser(SEQ ID NO: 4).

As the size of the peptide of the present invention becomes shorter, itsimmunogenicity inside the human body decreases while also being moreresistant to decomposition by proteases in the body, thus making itadvantageous.

In the above-mentioned amino acid sequence, Met indicates methionine,Leu leucine, Pro praline, Glu glutamate, Ser serine, Asp aspartate, Tyrtyrosine, Thr threonine and Phe phenylalanine, and all of said aminoacids are in the L form.

The above-mentioned amino acid sequence of the peptide of the presentinvention may contain replacement, deletion and/or insertion provided itis able to inhibit the phosphorylation of IκBα.

Those targets of substitution, deletion and/or insertion within the coresequence are the amino acids Tyr, Asp and Glu which are not targets ofphosphorylation. The number of targets of the amino acid is 1 to 2.However, it is preferable that the core amino acid sequence not bechanged by substitution, deletion and/or insertion. The number of aminoacids that can be changed by substitution, deletion and/or insertion inportions other than the core sequence may be 1 or more amino acids, andpreferably less than half, more preferably less than 30%, and even morepreferably less than 20%, for example 10% or less, of the total numberof amino acids other than the core portion of said peptide.

The peptide of the present invention may also be modified. Here,modification refers to modification of the structure of the amino acidsthemselves that compose the peptide, modification of the crosslinking orpeptide bonding between adjacent or non-adjacent amino acid residues,modification of the basic peptide backbone, introduction of side chainfunctional groups, ring formation and introduction of a spacer, etc.(Giannis, A. & Kolter, T.: Angrew. Chem. Ed. Engl. (1993) 32,1244-1267).

Salts of the above-mentioned peptide may be any salt provided it is apharmacologically acceptable acid addition salt or base addition salt.Examples of acid (inorganic acids and organic acids) addition saltsinclude inorganic acid addition salts such as hydrochlorides, hydrogenbromides, sulfates and nitrates, and organic acid addition salts such asacetates, benzoates, maleates, fumarates, succinates, tartrates,citrates, oxalates, metasulfonates, toluenesulfonates, aspartates andglutamates. Examples of base (inorganic bases and organic bases)addition salts include inorganic base addition salts such as sodiumsalts, potassium salts and calcium salts, and organic base additionsalts such as pyridine salts, triethylamine salts and lysine salts.

The peptide of the present invention can be produced by using methodsroutinely used in peptide synthesis, examples of which include liquidphase methods or solid phase methods (Peptide and Protein Drug Delivery,Lee, V. H. L. ed., Marcel Dekker, Inc. U.S.A.; PharmaceuticalBiotechnology, Borchardt, R. T. et al. ed., Prenum Press, New York andLondon). A starting material having a reactive carboxyl groupcorresponding to one of the two fragments that are designed by dividingat an arbitrary location of the peptide bond and synthesized by theabove-mentioned method, and a starting material having a reactive aminogroup corresponding to another fragment, are condensed using thecarboxyimide method or activated ester method and so forth, and in thecase the condensation product that is formed has a protective group,that protective group is then removed to produce the peptide of thepresent invention.

Those functional groups that are not to be involved in the reaction inthis reaction process are protected by protective groups. Examples ofprotective groups of amino groups include benzyloxycarbonyl,t-butyloxycarbonyl, p-biphenylisopropyloxycarbonyl and9-fluorenylmethyloxycarbonyl groups. Examples of protective groups ofcarboxyl groups include groups that are able to form alkyl esters orbenzyl esters and so forth. In the case of solid phase methods, however,the carboxyl group of the C-terminal is bonded to a carrier such aschlormethyl resin, oxymethyl resin or P-alkoxybenzylalcohol resin. Thecondensation reaction can be carried out in the presence of acondensation agent such as carboxyimide or an N-protected amino acidactivated ester or peptide activated ester can be used. Although theprotective groups are removed following completion of the condensationreaction, in the case of a solid phase method, the bond between theC-terminal of the peptide and the resin is also severed.

In addition, the peptide of the present invention can also be producedby using genetic engineering techniques (see the above-mentionedreferences Peptide and Protein Drug Delivery, and PharmaceuticalBiotechnology). For example, after synthesizing in accordance withroutine methods a oligonucleotide coding for a desired amino acidsequence among the nucleotide sequence of IκBα disclosed in WO 92-20795,and assembling by PCR, it is introduced into a suitable expressionvector. Next, suitable host cells are transformed with the expressionvector.

Peptide is produced in a culture liquid or host cells by culturing theresulting transformed cells under suitable conditions. A nucleotidesequence coding for FLAG peptide (see WO 88-04692) or humanimmunoglobulin constant region (see WO 94-28027, Ellison, J. et al., DNA(1981) 1, 11-18; Krawinkel, U. et al., EMBO J. (1982) 1, 403-407) may beadded to the nucleotide sequence coding for the peptide of the presentinvention, and it may be expressed in order to facilitate isolation andpurification of the peptide that is produced or increase its stability.

Any expression system can be used for producing the peptide of thepresent invention, examples of which include eucaryotic cells such asanimal cells including an established mammalian cell lines, fungalcells, yeast cells as well as procaryotic cells such as bacterial cellsincluding Escherichia coli. Preferably, the peptide of the presentinvention is expressed in mammalian cells, examples of which include COScells and CHO cells. In the case of using these cells, a routinely usedpromoter can be used that is useful for expressing in mammalian cells.For example, human cytomegalovirus immediate early promoter can be used.

Other promoters of gene expression in the mammalian cells that can beused for the present invention include viral promoters such as ofretrovirus, polio virus, adenovirus and simian virus 40 (SV40), andpromoters derived mammalian cells such as human polypeptide elongationfactor-1α (HEF-1α).

Replication origins that can be used include those derived SV40, poliovirus, adenovirus and bovine papilloma virus. Moreover, the expressionvector may contain as a marker phosphotransferase APH(3')II or I (neo)gene, thymidine kinase (TK) gene, E. coli xanthine guaninephosphoribosyl transferase (Ecogpt) gene or dihydrofolate reductase(DHFR) and so forth to increase the number of gene copies in the hostcells.

The peptide of the present invention is purified in accordance withroutine methods, examples of which include ion exchange chromatography,reverse phase liquid chromatography and affinity chromatography. Theamino acid sequence of peptides obtained in this manner can be analyzedwith a protein sequencer and identified by acid type analysis using anamino acid analyzer.

The peptide of the present invention comprises the IκBα phosphorylationsite and, provided it maintains the effect of inhibiting phosphorylationof IκBα, may contain replacement, deletion or insertion of amino acids.Moreover, its structure can also be modified to improve peptidestability, activity, half-life or cell membrane permeability (seeGiannis, A. & Kolter, T.: Angew. Chem. Int. Ed. Engl. (1993) 32,1244-1267). It is preferable that one modification of the peptide bemade to have cell membrane permeability.

For example, the peptide of the present invention can be given cellmembrane permeability by adding a lipolytic substance such as a steroidcompound or compound having a benzene ring to its terminal. Moreover,the peptide of the present invention can also be used by encapsulatingin a liposome to make it permeable to the cell membrane (see theabove-mentioned Peptide and Protein Drug Delivery, PharmaceuticalBiotechnology).

In addition, the peptide of the present invention can also be expressedin the body by synthesizing an oligonucleotide coding for the amino acidsequence of the peptide of the present invention in accordance withroutine methods, introducing it into a suitable expression vector inwhich it is able to be expressed, and administering into the body inaccordance with known techniques (see WO 94-27643, WO 92-20316, JapaneseUnexamined Patent Publication No. 6-303987, U.S. Pat. No. 5,166,320, WO92-19749 and WO 92-20316, etc.). Alternatively, the peptide of thepresent invention can also be used ex vivo in gene therapy. Namely, thepeptide of the present invention can be produced in transformed cellswithin the body by removing lymphocytes or other blood cells from thebody, transforming these cells with the above-mentioned expressionvector, and returning the transformed cells to the body (see JapaneseUnexamined Patent Publication No. 6-329559).

Since the peptide of the present invention has the effect of inhibitingphosphorylation of IκBα, it is considered to inhibit the expression ofinflammatory cytokine and inflammatory mediator genes as well as therelease of their gene products. It is therefore effective in thetreatment and prevention of inflammatory diseases of humans and othermammals and can also be used as an immunosuppressant. Moreover, sincethe peptide of the present invention is directed to the inhibition ofNFκB activation by being targeted at inhibition of the phosphorylationof IκBα present in cells, its effects are surmised to be specific.

By using the peptide of the present invention as an antiphlogistic, itis expected to be useful in the treatment of various inflammatorydiseases such as multiple sclerosis, systemic lupus erythematosus,articular rheumatism, psoriasis, gout, nephritis, inflammatory colitis,myocardial infarction, asthma, Mediterranean fever, Crohn's disease,adult respiratory distress syndrome, pulmonary emphysema, cysticfibrosis, hypersensitive pneumonia, tuberculous hydrothorax,carcinomatous hydrothorax, sarcoidosis, idiopathic fibroid lung, diffusepanbronchiolitis, following esophageal cancer surgery and ischemicreperfusion syndrome. In addition, the peptide of the present inventionis also effective in the suppression of rejection reactions during bonemarrow transplantation, organ transplantation and so forth by using asan immunosuppressant.

Although the peptide of the present invention may be administered byoral administration or parenteral administration, parenteraladministration is particularly preferable. Examples of parenteraladministration include injection, intrarectal administration,percutaneous administration and transpulmonary administration. Althoughthe dose of the peptide of the present invention varies according to theadministration method, patient symptoms, age and so forth, it isnormally administered over the course of 1 to 4 administrations per dayat 0.001 to 1000 mg, and preferably 0.01 to 10 mg, per administration.

The peptide of the present invention is normally administered in theform of a preparation formulated by mixing with a carrier or vehicle forpharmaceutical preparations (Remington's Pharmaceutical Science, LatestEdition, Mark Publishing Company, Easton, U.S.A.). Examples ofpreparation carriers and vehicles are those which are routinely used inthe preparation field of pharmaceuticals, and substances that do notreact with the peptide of the present invention. For example, whenproducing an injection preparation, pH adjusting agent such ashydrochloric acid, sodium hydroxide, sodium lactate, lactic acid, sodiumhydrogen phosphate and sodium dihydrogen phosphate, isotonic agents suchas sodium chloride and glucose, and the active ingredient in the form ofthe peptide of the present invention are dissolved as necessary inphysiological saline for injection, and either filled into ampulesfollowing sterilization filtration, or freeze-dried in a vacuum afteradding mannitol, dextrin, cyclodextrin, gelatin and so forth to preparean injection preparation that is reconstituted at the time of use.

Alternatively, an emulsion for injection can also be prepared by addinglecithin, polysorbitol 80, polyoxyethylene hardened palm oil and soforth and emulsifying in water. When producing an intrarectalpreparation, the active ingredient in the form of the peptide of thepresent invention along with a suppository base such as cacao butter,tri-, di- or monoglycerides of fatty acids or polyethyleneglycol aremoisturized and dissolved, and poured into a mold followed either bycooling or coating with a gelatin film dissolved in polyethylene glycolor soy bean oil and so forth.

When producing a percutaneous preparation, white Vaseline, bees wax,liquid paraffin or polyethylene glycol and so forth are added to theactive ingredient in the form of the peptide of the present invention asnecessary followed by moisturizing and mixing to form an ointment, orafter mixing with an adhesive such as rosin or alkylester acrylatepolymer and so forth, spreading over a non-woven fabric of polyethyleneand so forth to form a tape.

When manufacturing a transpulmonary preparation, the active ingredientin the form of the peptide of the present invention is dissolved ordispersed in an ordinary spray and filled into a pressurized containerto form an aerosol.

Moreover, the peptide of the present invention can be prepared in theform of a continuous preparation by using known liposome technology andso forth.

In addition, the above-mentioned preparations may also contain otheringredients that are effective in the treatment of the target disease.

The following provides a detailed explanation of the present inventionby describing Examples. However, the present invention is not limited tothese Examples.

EXAMPLES Reference Example 1 Preparation of Glutathione S-transferase(GST)-IκBα Fused Protein

(1) Construction of DNA Coding for GST-IκBα Fused Protein

In order to isolate the cDNA of human IκBα, specific primers GMD5 (SEQID NO: 5) and GMD3 (SEQ ID NO: 6) were synthesized with anoligonucleotide synthesizer for each of the nucleotide sequences in thevicinity of the start codon and stop codon, respectively, of IκBα cDNAbased on the report of Haskill, S. et al. (Cell (1991) 65, 1281-1289).Furthermore, the primers were designed so that a site cleaved withrestriction enzyme EcoRI was inserted in the 5'-end of primer GMD5, anda site cleaved with restriction enzyme BamHI was inserted in the 5'-endof primer GMD3. Total RNA was obtained from lipopolysaccharide(LPS)-stimulated human peripheral blood mononuclear cells using RNA zolB(Biotex Laboratory, Houston, U.S.A.).

Using the resulting total RNA as a template, reverse transcriptase-PCR(polymerase chain reaction) was performed using the above-mentionedprimers GMD5 and GMD3 to obtain the entire length of human IκBα cDNA.The resulting entire length of human IκBα cDNA was introduced at theEcoRI and BamHI cleavage sites of plasmid vector pGENT2 containingglutathione S-transferase (GST) gene (Murakami, S. et al.: J. Biol.Chem. (1994) 269, 15118-15123) to obtain plasmid pGST-MD. The nucleotidesequence of the entire length of human IκBα cDNA was confirmed using themethod of Sanger, F. et al. (Proc. Natl. Acad. Sci., USA (1977) 74,5463-5467). A schematic drawing of plasmid pGST-MD is shown in FIG. 1.

(2) Construction of DNA Coding for GST-Partial Deletion IκBα MutantFused Proteins

DNAs coding for six fused proteins comprising GST and six partialdeletion mutants of IκBα were constructed using synthetic primers. Eachprimer had a nucleotide sequence corresponding to the respectivedeletion site. Primer GMD5 (SEQ ID NO: 5) and primer GMD6 (SEQ ID NO: 7)were designed so as to define each 5'-end and 3'-end of the DNA codingfor mutant del-1 consisting of amino acids 1 to 242 of human IκBα.Primer GMD5 (SEQ ID NO: 5) and primer GMD7 (SEQ ID NO: 8) were designedso as to define each 5'-end and 3'-end of the DNA coding for mutantdel-2 consisting of amino acids 1 to 181 of human IκBα.

Primer GMD52 (SEQ ID NO: 9) and primer GMD3 (SEQ ID NO: 6) were designedso as to define each 5'-end and 3'-end of the DNA coding for mutantdel-4 consisting of amino acids 73 (1st ankirin repeat) to 317 of humanIκBα. Primer GMD53 (SEQ ID NO: 10) and primer GMD3 (SEQ ID NO: 6) weredesigned so as to define each 5'-end and 3'-end of the DNA coding formutant del-5 consisting of amino acids 182 (3rd ankirin repeat) to 317of human IκBα.

Primer GMD5 (SEQ ID NO: 5) and primer GMD9 (SEQ ID NO: 11) were designedso as to define each 5'-end and 3'-end of the DNA coding for mutantdel-9 consisting of amino acids 1 to 295 of human IκBα. Primer GMD5 (SEQID NO: 5) and primer GMD10 (SEQ ID NO: 12) were designed so as to defineeach 5'-end and 3'-end of the DNA coding for mutant del-10 consisting ofamino acids 1 to 282 of human IκBα.

Furthermore, each primer was designed so that a site cleaved byrestriction enzyme EcoRI or BamHI was introduced on the 5'-end. Usingplasmid vector pGENT2 containing the entire length of human IκBα cDNA asa template, PCR was performed using each primer pair that defines the5'-end and 3'-end. The resulting DNA coding for each mutant wasintroduced into EcoRI and BamHI cleaved sites of plasmid vector pGENT2.The nucleotide sequence of each mutant cDNA was confirmed using theabove-mentioned method of Sanger, F. et al. The structure of each mutantis schematically illustrated in FIG. 2.

(3) Construction of DNA Coding for GST-Substituted IκBα Mutant

DNA was constructed that codes for the fused protein consisting of GSTand IκBα-mutant K (mt K) having a substitution mutation of the aminoacids in the acidic region of the C-terminus of IκBα.

Using the animal cell expression vector, PRC/CMV, containing DNA codingfor IκBα-mt K in which serine at position 283, serine at position 288,threonine at position 291, serine at position 293, threonine at position296 and threonine at position 298 of the acidic region of the C-terminusare all substituted with analine (Ernst, M. K. et al.: Mol. Cell. Biol.(1995) 872-882) for the template, PCR was performed using specificprimers GMD5 and GMD3 for the nucleotide sequences in the proximity ofthe start codon and stop codon of IκBα cDNA. The resulting DNA codingfor IκBα substituted mutant-mt K was introduced into the sites cleavedwith EcoRI and BamHI of plasmid vector pGENT2. The nucleotide sequenceof the cDNA of mutant mt K was confirmed using the above-mentionedmethod of Sanger. F. et al. A schematic drawing of the acidic region ofIκBα-mutant K is shown in FIG. 3.

(4) Preparation of GST-IκBα Fused Protein

Each of the expression vectors obtained in the above-mentioned steps(1), (2) and (3) were expressed in Escherichia coli to obtain GST-IκBαfused proteins.

After transforming Escherichia coli (JM109) with each expression vector,the microorganisms were cultured in 2×YT culture medium (Wako PureChemical Industries, Ltd.) containing 50 μg/ml of ampicillin. Themicroorganisms were stimulated by addition of 1 mMisopropylthiogalactoside (IPTG, Wako Pure Chemical Industries, Ltd.)after which the microorganisms were collected 2 to 3 hours later.

After separating by centrifugation, the pellet was washed with PBS(-)(Nissui Pharmaceutical Co., Ltd.). After pulverizing the pellet withultrasonic waves in PBS(-) containing 1% Triton X-100 (Wako PureChemical Industries, Ltd.), the suspension was separated bycentrifugation and the supernatant was recovered. Next, each fusedprotein was bound to glutathione sepharose (Pharmacia) equilibrated withPBS(-) containing 1% Triton X-100, and after washing five times withPBS(-), was eluted with 5 mM glutathione solution (Wako Pure ChemicalIndustries, Ltd.) (Smith, D. B. & Johnson, K. S.: Gene (1986) 67,31-40).

Reference Example 2 Partial Purification of IκBα Phosphorylase (Kinase)

The kinase that phosphorylates IκBα was partially purified from humanmonocytic cell line THP-1. THP-1 cells were cultured at 37° C. and inthe presence of 5% CO₂ in RPMI-1640 culture medium (Gibco BRL)containing 5% fetal calf serum (FCS, ICN Corp., Australia), 100 U/mlpenicillin (Gibco BRL), 100 μg/ml streptomycin (Gibco BRL) and 2 mML-glutamine (Wako Pure Chemical Industries, Ltd.). 4×10⁹ THP-1 cellswere stimulated for 5 minutes with 10 μg/ml LPS (E. coli 055-B5, Difco)and collected by centrifugation.

After washing the cell sediment twice with PBS(-), the sediment wassuspended in 20 mM Hepes buffer (pH 7.9) containing 0.5 mMphenylmethylsulfonylfluoride (PMSF) and 1 μg/ml each of aprotinin,leupeptin and pepstatin (Sigma) followed by ultrasonic pulverization andcentrifugation for 10 minutes at 500×g. The supernatant was furthercentrifuged for 35 minutes at 4° C. and 18000×g and dialyzed for 4 hoursat 4° C. against 20 mM Tris buffer (pH 7.5) to obtain the crudefraction.

Next, the crude fraction was separated by elution by increasing the NaClconcentration in stepwise fashion at 0 M, 0.5 M and 2 M using a Red-Asepharose column (Amicon, bed volume: 5 ml) equilibrated with 20 mM Trisbuffer (pH 7.5). The fraction eluted with 2 M NaCl was dialyzed for 4hours at 4° C. against 20 mM Tris buffer (pH 7.5). This fraction wasseparated by elution by increasing the NaCl concentration in stepwisefashion at 0 M, 0.1 M, 0.25 M, 0.5 M and 2 M using a dimethylaminoethyl(DEAE) sephacel column (Pharmacia, bed volume: 20 ml) equilibrated with20 mM Tris buffer (pH 7.5). After dialyzing the fraction eluting at 0.25M NaCl for 4 hours at 4° C. against 20 mM Tris buffer (pH 7.5), theresulting fraction was eluted at the rate of 1 ml/min with DEAE-HPLC(DEAE-5PW, Tosoh) equilibrated with 20 mM Tris buffer (pH 7.5).

Fractions were collected in 2 ml aliquots while applying a linearconcentration gradient from 0 to 0.5 M NaCl from 10 to 70 minutes afterthe start of elution. The activity of the resulting kinase was assayedusing in vitro kinase assay (see Embodiment 1(1)) in the solid phaseusing glutathione sepharose coupled with GST-IκBα. The amount of proteinwas assayed using Coomassie Protein Reagent (Pierce) and bovine serumalbumin (BSA) for the standard. As a result, the resulting partiallypurified IκBα kinase was purified by a factor of approximately 126 times(see Table 1).

    ______________________________________                                                  Protein            Specific                                            content Activity activity Purification                                       Step (mg) (×10.sup.-6 cpm) (×10.sup.-6 cpm) (times)             ______________________________________                                        Crude     126.16   104.4     0.83    1                                          fraction                                                                      Red-A 9.73 66.7 6.86 8.26                                                     Sepharose                                                                     (0.5-2M                                                                       NaCl)                                                                         DEAE-sephacel 4.54 64.3 14.16 17.6                                            (0.1-0.25M                                                                    NaCl)                                                                         DEAE-HPLC 0.41 41.7 104.24 125.60                                             (fr. 17-20)                                                                 ______________________________________                                    

Example 1 Measurement of Phosphorylation Activity Against IκBα

(1) Experiment Using GST-IκBα Fused Protein

The phosphorylation activity against IκBα was measured by in vitrokinase assay. Each of the fused proteins prepared in Reference Example 1and glutathione Sepharose beads (Pharmacia) were coupled by stirring for2 hours at 4° C. and suspended in PBS(-) after washing three times withPBS(-) containing 1% NP-40 (Nakarai-Tesk) and twice with PBS(-). 20 μlof glutathione Sepharose beads containing 14 pmoles of fused protein,and 2 μl of purified IκBα kinase derived from the cytoplasm fractionobtained in the above-mentioned Reference Example 2 were prepared to 400μl with PBS(-) followed by stirring for 3 hours at 4° C.

Next, precipitate was obtained by washing twice with PBS(-) containing1% NP-40 and once with PBS(-). 5 μCi (185 kBq) of [γ-32P]ATP (AmershamJapan, #PB10168, 3000 Ci/mM) and 20 μl of kinase buffer (20 mM Hepesbuffer containing 20 mM MgCl₂ and 10 mM MnCl₂ (pH 7.4)) were added tothis precipitate and incubated for 5 minutes at 30° C. The reaction wasstopped by adding PBS(-) containing 13.5 mM EDTA and 1% NP-40 followedby washing four times with PBS(-) containing 1% NP-40, adding 5 μl ofsodium dodecyl sulfate (SDS) sample buffer (0.0625 M Tris-HCl (pH 6.8),10% (w/v) glycerol, 5% (w/v) β-mercaptoethanol, 2.3% (w/v) SDS), andeluting protein in boiling water.

After separating by centrifugation, the supernatant was applied onSDS-polyacrylamide electrophoresis (PAGE). Following completion ofSDS-PAGE, autoradiography was performed using X-ray film (see FIG. 4).As a result, although radioactivity was detected in the cases of usingfused proteins with GST of the entire length of IκBα (wild type; wt),mutant del-9, del-5 or del-4, radioactivity was not detected in the caseof using fused proteins of GST and mutant del-10 or del-1.

In addition, in a portion of the experiment, the above-mentioned kinasebuffer was added to 14 pmoles of fused protein and 2 μl of partiallypurified IκBα kinase derived from the cytoplasm fraction of THP-1 cellswithout using glutathione Sepharose beads so that the finalconcentrations were equal after which assay was performed as describedabove with the liquid phase (see FIG. 5). As a result, althoughradioactivity was detected in the cases of using each fused protein ofGST and the entire length of IκBα (wt) and del-9, radioactivity was notdetected in the cases of using fused proteins of GST and mutant del-10,del-1 and substitution mutant (mt-K). These results clearly showed thatthe phosphorylation site exists in a region in which the amino acidsequences of mutants del-10 and of del-9 differ, and that IκBα is notphosphorylated when amino acid substitution is present in substitutionmutant mt K.

(2) Competitive Inhibition of Phosphorylation Activity by Peptide

A peptide of the IκBα phosphorylation site having the amino acidsequence of SEQ ID NO: 1 was synthesized using a peptide synthesizer andpurified by reverse phase HPLC after which its sequence was confirmedusing an amino acid analyzer. The phosphorylation activity of IκBα wasthen assayed using this peptide.

An equal amount of the above-mentioned purified IκBα kinase derived fromthe cytoplasm fraction of THP-1 cells was added to fused protein of GSTand IκBα (wt) followed by the addition of 2.7 nM of peptide (200 timesthe molar ratio) and assaying in the same manner as in (1) above. Thoseresults are shown in FIG. 6. Phosphorylation of IκBα was inhibited bythe addition of peptide of the IκBα phosphorylation site.

(3) Identification of Phosphorylation Site

In order to further identify the phosphorylation site of IκBαphosphorylation inhibitory peptide, in vitro kinase assay was performedusing peptides in which one amino acid residue was substituted in theIκBα phosphorylation inhibitory peptide (SEQ ID NO: 1) prepared inExample 1(2).

An assay was performed in a liquid phase using the same conditions as inthe in vitro kinase assay of Example 1(2) using 15 nmoles each ofpeptides in which serine at position 283, serine at position 288,threonine at position 291 or serine at position 293, which are expectedto be the amino acid residues that are phosphorylated in the amino acidsequence of IκBα phosphorylation inhibitory peptide having the aminoacid sequence of SEQ ID NO: 1, was substituted with one alanine residueeach (283 Ala (SEQ ID NO: 13), 288 Ala (SEQ ID NO: 14), 291 Ala (SEQ IDNO: 15) or 293 Ala (SEQ ID NO: 16), respectively) (n=4). Furthermore, apositive control in which the entire length of IκBα (wt) was added and anegative control in which IκBα was not added were used as controls.

Following the assay, SDS polyacrylamide gel electrophoresis wasperformed in the same manner as Example 1(1) using Tris/Tricine buffer.The results were analyzed and quantified with an image analyzer(BAS1000, Fuji Film Co., Ltd.). Those results are shown in FIG. 7. As aresult, peptide 283 Ala did not have an effect on phosphorylation. Onthe other hand, peptide 293 Ala had a significant effect onphosphorylation. In addition, peptides 288 Ala and 291 Ala only had aslight effect on phosphorylation.

Based on the above results, it was strongly suggested that serine atposition 288, threonine at position 291 and serine at position 293 ofthe four serine or threonine residues in the amino acid sequence of IκBαphosphorylation inhibitory peptide are participated in thephosphorylation.

Furthermore, the reason why phosphorylation was not completely inhibitedwhen each of the above-mentioned IκBα substituted mutants was used isthought to be because a serine or threonine residue other than thesubstituted amino acid residue was phosphorylated.

Example 2 Effect of Peptide of the IκBα Phosphorylation Site on NFκBActivity

The effect on NFκB activation in a cell-free system (Ishikawa, Y. etal., J. Biol. Chem. (1995) 362, 79-83) was investigated using peptidesof the IκBα phosphorylation site.

Non-stimulated THP-1 cells were cultured under the conditions describedin Reference Example 2. The cytoplasmic fraction and plasma membraneenriched fraction were prepared from THP-1 cells in accordance with themethod of Sadowski, H. B. et al. (Nature (1993) 362, 79-83). Both 20 μgfractions were combined followed by the addition of 15 nM IκBαphosphorylation site peptide, 10 mM ATP and 20 μg/ml of LPS andstimulation for 10 minutes at 30° C.

After stopping the reaction by adding 13.5 mM EDTA (pH 8.0), thesupernatant was obtained by centrifugal separation. Gel shift assay wasperformed on this supernatant using a ³² P-labeled DNA probe containingthe IL-8κB binding site in 0.25×TBE (final concentrations: 13.5 g Trissalt (Sigma), 6.9 g boric acid (Wako Pure Chemical Industries, Ltd.) and1.165 g EDTA (Wako Pure Chemical Industries, Ltd.) per liter) containing6% polyacrylamide gel (Acrylamide: bis=30:1) (Mukaida, N. et al., J.Biol. Chem. (1994) 269, 13289-13295). This was followed by analysis andquantification with an image analyzer (BAS1000, Fuji Film Co., Ltd.).

Furthermore, a control group to which LPS was not added, and groups towhich were added 15 nM each of casein kinase 11 substrate (Kaengel, E.A. et al., J. Biol. Chem. (1987) 262, 9136-9140) (SEQ ID NO: 17) and MAPkinase substrate (Dong, Z. et al., J. Exp. Med. (1993) 177, 1071-1077)(SEQ ID NO: 18) were used.

The results of gel shift assay are shown in FIG. 8. In the system inwhich THP-1 cells stimulated with LPS were used, phosphorylation of IκBαwas inhibited in the group to which was added IκBα phosphorylation sitepeptide, and as a result, NFκB activity was not observed. Moreover,analysis and quantification results with the image analyzer are shown inFIG. 9. NFκB activity was clearly inhibited in the group to which wasadded IκBα phosphorylation site peptide.

Industrial Applicability

According to the present invention, an antiinflammatory agent andimmunosuppressant are provided that are effective in treatment ofdiseases involving inflammatory cytokines and inflammatory mediatorsincluding inflammatory diseases such as multiple sclerosis, systemiclupus erythematosus, and rejection reactions during bone marrowtransplantation and organ transplantation.

The peptide or its salt contained as an active ingredient of theantiinflammatory agent and immunosuppressant provided by the presentinvention inhibits phosphorylation of IκBα, which as a result ofinhibiting the effect of transcription activation by NFκB, is able toalleviate the above-mentioned diseases by administering to patientssuffering from said diseases.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 20                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -      Met Leu Pro Glu Ser Glu Asp Glu - # Glu Ser Tyr Asp Thr Glu       Ser Glu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Phe Thr Glu Phe Thr Glu Asp Glu - # Leu                                              20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -      Met Leu Pro Glu Ser Glu Asp Glu - # Glu Ser Tyr Asp Thr Glu       Ser Glu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Phe                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -      Ser Glu Asp Glu Glu Ser Tyr Asp - # Thr Glu Ser Glu Phe                 1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -      Ser Tyr Asp Thr Glu Ser                                                  1             - #  5                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "Synthetic DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GCGAATTCCA TGTTCCAGGC GGCCGAGCG         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "Synthetic DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GCAGGATCCT CATAACGTCA GACGCTGGC         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "Synthetic DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - GCAGGATCCT CAATCAGCCC CACACTTCAA CAG       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "Synthetic DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GCAGGATCCT CAGTAGTTGG TAGCCTTCAG GAT       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "Synthetic DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - GCGAATTCCG ACGGGGACTC GTTCCTGCA         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "Synthetic DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - GCGAATTCCA ATGGCCACAC GTGTCTACAC         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "Synthetic DNA"                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - GCAGGATCCT CAGAACTCTG ACTCTGTGTC ATA       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "Sythetic DNA"                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - GCAGGATCCT CACTCTGGCA GCATCTGAAG GTT       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - -      Met Leu Pro Glu Ala Glu Asp Glu - # Glu Ser Tyr Asp Thr Glu        Ser Glu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Phe Thr Glu Phe Thr Glu Asp Glu - # Leu                                              20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - -      Met Leu Pro Glu Ser Glu Asp Glu - # Glu Ala Tyr Asp Thr Glu       Ser Glu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Phe Thr Glu Phe Thr Glu Asp Glu - # Leu                                              20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - -      Met Leu Pro Glu Ser Glu Asp Glu - # Glu Ser Tyr Asp Ala Glu       Ser Glu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Phe Thr Glu Phe Thr Glu Asp Glu - # Leu                                              20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - -      Met Leu Pro Glu Ser Glu Asp Glu - # Glu Ser Tyr Asp Thr Glu       Ala Glu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Phe Thr Glu Phe Thr Glu Asp Glu - # Leu                                              20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - -      Arg Arg Arg Glu Glu Glu Thr Glu - # Glu Glu                              1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - -      Ala Pro Arg Thr Pro Gly Gly Arg - # Arg                                  1             - #  5                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - -      Met Leu Pro Glu Ser Glu Asp Glu - # Glu Ser Tyr Asp Thr Glu       Ser Glu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Phe Thr Glu Phe Thr Phe Asp Glu - # Leu Pro Tyr Asp Asp                             20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - -      Met Leu Pro Glu Ala Glu Asp Glu - # Glu Ala Tyr Asp Ala Glu        Ala Glu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Phe Ala Phe Phe Ala Glu Asp Glu - # Leu                                              20 - #                 25                                  __________________________________________________________________________

We claim:
 1. A peptide which inhibits phosphorylation activity of humanIκBα and consists of the amino acid sequence of SEQ ID NO: 1, or apharmaceutically acceptable salt thereof.
 2. A peptide which inhibitsphosphorylation activity of human IκBα and consists of the amino acidsequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.3. A peptide which inhibits phosphorylation activity of human IκBα andconsists of the amino acid sequence of SEQ ID NO: 3, or apharmaceutically acceptable salt thereof.
 4. A peptide which inhibitsphosphorylation activity of human IκBα and consists of the amino acidsequence of SEQ ID NO: 4, or a pharmaceutically acceptable salt thereof.5. A peptide which inhibits phosphorylation activity of human IκBα,which is a fragment of the amino acid sequence of SEQ ID NO: 1 and whichcomprises the amino acid sequence of SEQ ID NO: 4, or a pharmaceuticallyacceptable salt thereof.
 6. A peptide which inhibits phosphorylationactivity of human IκBα, which is a fragment of an amino acid sequencemodified from the amino acid sequence of SEQ ID NO: 1, and whichcomprises an amino acid sequence of SEQ ID NO: 4, wherein saidmodification from the amino acid sequence of SEQ ID NO: 1 is areplacement and/or deletion of one or more amino acids in SEQ ID NO: 1other than the amino acids in SEQ ID NO: 4, or a pharmaceuticallyacceptable salt thereof.
 7. A composition for inhibition ofphosphorylation activity of human IκBα, comprising a peptide accordingto claim
 1. 8. A composition for inhibition of phosphorylation activityof human IκBα, comprising a peptide according to claim
 2. 9. Acomposition for inhibition of phosphorylation activity of human IκBα,comprising a peptide according to claim
 3. 10. A composition forinhibition of phosphorylation activity of human IκBα, comprising apeptide according to claim
 4. 11. A composition for inhibition ofphosphorylation activity of human IκBα, comprising a peptide accordingto claim
 5. 12. A composition for inhibition of phosphorylation activityof human IκBα, comprising a peptide according to claim
 6. 13. Animmunosuppressant composition comprising a peptide according to claim 1.14. An immunosuppressant composition comprising a peptide according toclaim
 2. 15. An immunosuppressant composition comprising a peptideaccording to claim
 3. 16. An immunosuppressant composition comprising apeptide according to claim
 4. 17. An immunosuppressant compositioncomprising a peptide according to claim
 5. 18. An immunosuppressantcomposition comprising a peptide according to claim
 6. 19. Animmunosuppressant composition comprising a peptide or a salt thereofaccording to claim
 1. 20. An immunosuppressant composition comprising apeptide or a salt thereof according to claim
 2. 21. An immunosuppressantcomposition comprising a peptide or a salt thereof according to claim 3.22. An immunosuppressant composition comprising a peptide or a saltthereof according to claim
 3. 23. An immunosuppressant compositioncomprising a peptide or a salt thereof according to claim
 4. 24. Animmunosuppressant composition comprising a peptide or a salt thereofaccording to claim 5.